Journal: Experimental and Therapeutic Medicine

Article Title: miR-382-3p downregulation contributes to the carcinogenesis of lung adenocarcinoma by promoting AKT SUMOylation and phosphorylation

doi: 10.3892/etm.2022.11367

Figure Lengend Snippet: Expression of SAE1, a target of miR-382-3p, is negatively correlated with miR-382-3p levels in tumors. (A) Gene Ontology analysis of predicted miR-382-3p targets. (B and C) SAE1 protein levels in tumor and adjacent non-tumor control samples were examined by immunoblotting. (B) Representative western blots and (C) comparison of SAE1 protein levels between tumor and adjacent non-tumor control samples for each case. (D) Correlation analysis of the SAE1 protein levels and miR-382-3p levels in lung adenocarcinoma tumor samples. (E) Kaplan-Meier curves depicting the overall survival of 1,044 patients with lung cancer. Data were from Kaplan-Meier Plotter. SAE1, small ubiquitin-like modifier 1 activating enzyme subunit 1; miR, microRNA; MF, molecular function; CC, cellular component; BP, biological process; T, tumor tissue; N, normal tissue.

Article Snippet: The following antibodies were used: Rabbit anti-SAE1 polyclonal antibody (cat no. 13585S; 1:1,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-SUMO1 polyclonal antibody (cat no. 4930S; 1:2,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-pAkt (Ser473) polyclonal antibody (cat no. 4060S; 1:2,000 dilution; Cell Signaling Technology, Inc.), mouse anti-GAPDH monoclonal antibody (cat no. sc-47724; 1:2,000 dilution; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Control, Western Blot, Comparison, Ubiquitin Proteomics

Journal: Experimental and Therapeutic Medicine

Article Title: miR-382-3p downregulation contributes to the carcinogenesis of lung adenocarcinoma by promoting AKT SUMOylation and phosphorylation

doi: 10.3892/etm.2022.11367

Figure Lengend Snippet: miR-382-3p represses SAE1 expression directly by targeting its 3'UTR. (A) Dual-luciferase assay. (B) miR-382-3p mimics or inhibitor were transfected into A549 and H1299 cells. At 48 h after transfection, the cells were subjected to protein exaction and immunoblotting. (C) The levels of SAE1 and Ki-67 in xenograft tumors generated from miR-382-3p-overexpressing A549 and H1299 cells were examined by immunohistochemistry (scale bars, 50 µM). * P<0.05, ** P<0.01. SAE1, small ubiquitin-like modifier 1 activating enzyme subunit 1; miR, microRNA; WT, wild-type; Mut, mutant.

Article Snippet: The following antibodies were used: Rabbit anti-SAE1 polyclonal antibody (cat no. 13585S; 1:1,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-SUMO1 polyclonal antibody (cat no. 4930S; 1:2,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-pAkt (Ser473) polyclonal antibody (cat no. 4060S; 1:2,000 dilution; Cell Signaling Technology, Inc.), mouse anti-GAPDH monoclonal antibody (cat no. sc-47724; 1:2,000 dilution; Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Luciferase, Transfection, Western Blot, Generated, Immunohistochemistry, Ubiquitin Proteomics, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: miR-382-3p downregulation contributes to the carcinogenesis of lung adenocarcinoma by promoting AKT SUMOylation and phosphorylation

doi: 10.3892/etm.2022.11367

Figure Lengend Snippet: miR-382-3p inhibition upregulates AKT SUMOylation and activates the AKT signaling pathway. (A) Immunoblotting was used to detect the levels of full-scale SUMOylation, SUMO1, AKT and p-AKT in tumor and non-tumor control samples. (B) A549 and H1299 cells were transfected with miR-382-3p inhibitor for 48 h. The cells were then subjected to RT-qPCR and immunoblotting. (C) The expression of AKT signaling downstream genes was examined by RT-qPCR. * P<0.05, ** P<0.01, *** P<0.001 vs. control. miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; p-AKT, phosphorylated AKT; T, tumor tissue; N, normal tissue; IP, immunoprecipitation; IB, immunoblot; CCND1, cyclin D1; CDK2, cyclin-dependent kinase 2; SUMO1, small ubiquitin-like modifier 1.

Article Snippet: The following antibodies were used: Rabbit anti-SAE1 polyclonal antibody (cat no. 13585S; 1:1,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-SUMO1 polyclonal antibody (cat no. 4930S; 1:2,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-pAkt (Ser473) polyclonal antibody (cat no. 4060S; 1:2,000 dilution; Cell Signaling Technology, Inc.), mouse anti-GAPDH monoclonal antibody (cat no. sc-47724; 1:2,000 dilution; Santa Cruz Biotechnology, Inc.).

Techniques: Inhibition, Western Blot, Control, Transfection, Quantitative RT-PCR, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunoprecipitation, Ubiquitin Proteomics

Journal: Experimental and Therapeutic Medicine

Article Title: miR-382-3p downregulation contributes to the carcinogenesis of lung adenocarcinoma by promoting AKT SUMOylation and phosphorylation

doi: 10.3892/etm.2022.11367

Figure Lengend Snippet: miR-382-3p inhibition promotes proliferation and inhibits apoptosis in lung adenocarcinoma cell lines. A549 and H1299 cells were transfected with miR-382-3p inhibitor, with or without SAE1-specific siRNA for 48 h. (A) The cells were subjected to MTT assays to examine proliferation and viability. (B) Flow cytometric analysis was used to detect cell viability and apoptosis. * P<0.05, ** P<0.01. (C and D) Cell cycle analysis of (C) A549 and (D) H1299 cells. * P<0.05 S phase vs. inhibitor control; # P<0.05, ## P<0.01 G2-M phase vs. inhibitor control. ns, no significance; miR, microRNA; PI, propidium iodide; APC, allophycocyanin; siRNA, small interfering RNA; si-SAE1, siRNA targeting SAE1; SAE1, small ubiquitin-like modifier 1 activating enzyme subunit 1; FL-H, fluorescence-height.

Article Snippet: The following antibodies were used: Rabbit anti-SAE1 polyclonal antibody (cat no. 13585S; 1:1,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-SUMO1 polyclonal antibody (cat no. 4930S; 1:2,000 dilution; Cell Signaling Technology, Inc.), rabbit anti-pAkt (Ser473) polyclonal antibody (cat no. 4060S; 1:2,000 dilution; Cell Signaling Technology, Inc.), mouse anti-GAPDH monoclonal antibody (cat no. sc-47724; 1:2,000 dilution; Santa Cruz Biotechnology, Inc.).

Techniques: Inhibition, Transfection, Cell Cycle Assay, Control, Small Interfering RNA, Ubiquitin Proteomics, Fluorescence